Human hair is principally composed of hair keratins and keratin-associated proteins (KAPs) that form a complex network giving the hair its rigidity and mechanical properties. However, during their growth, hairs are subject to various treatments that can induce irreversible damage. For a better understanding of the human hair protein structures, proteomic mass spectrometry (MS)-based strategies could assist in characterizing numerous isoforms and posttranslational modifications of human hair fiber proteins. However, due to their physicochemical properties, characterization of human hair proteins using classical proteomic approaches is still a challenge. To address this issue, we have used two complementary approaches to analyze proteins from the human hair cortex. The multidimensional protein identification technology (MudPit) approach allowed identifying all keratins and the major KAPs present in the hair as well as posttranslational modifications in keratins such as cysteine trioxidation, lysine, and histidine methylation. Then two-dimensional gel electrophoresis coupled with MS (2-DE gel MS) allowed us to obtain the most complete 2-DE gel pattern of human hair proteins, revealing an unexpected heterogeneity of keratin structures. Analyses of these structures by differential peptide mapping have brought evidence of cleaved species in hair keratins and suggest a preferential breaking zone in α-helical segments.

Keywords:

ageing;

hair keratin;

hair structure;

keratin-associated proteins

Abstract

Abstract: In many cultures, a youthful look is strictly linked to strong and healthy hair. Source of the hair fibre is the hair follicle, a highly specialized skin appendage. Biological alterations because of intrinsic or extrinsic stimuli can destabilize this perfectly organized system, thus effecting hair growth or metabolism. Also, ageing could be characterized as a disturbance in this well-balanced machinery. Albeit the predominant symptom of hair ageing, greying, is addressed in a plurality of research activities, further age-related changes, e.g. related to hair structure, remain obscure. Therefore, we characterized hair follicles of two volunteer panels (below 25 years, above 50 years) on the molecular level, especially focussing on alterations influencing gene expression of keratins and keratin-associated proteins. We showed that concordantly to other biological systems the hair follicle undergoes several modifications during the ageing process associated among others with a significant decline in these structural proteins. Providing strategies to fight against these age-related changes is a challenge for hair science.

Background

Since ancient times, the human hair is an attribute for health, youth and attractiveness and plays an important role in people’s self-perception (1). Therefore, alterations in the appearance of hair, such as premature greying or changes in hair structure, often stress the well-being and self-confidence of the affected persons.

The hair follicle is a complex mini organ that shows cyclic activity during postnatal life with periods of active growth, involution and resting (2). In each anagen phase, it produces the visible hair shaft; thus, synthesis of hair keratin is an essential prerequisite for the growth of strong and healthy hair. But like all biological systems, the hair follicle, the biological active part of the hair, also undergoes an ageing process, which is not only characterized by loss of pigmentation (3–6). But even if there are some general features besides greying indicating follicular ageing, such as hair loss, reduction in hair diameter as well as anecdotal evidence that hair becomes more fragile at an older age, little is known about further alterations and the molecular reasons underlying the known macroscopic changes.

Questions addressed

The objective of this study was to characterize the age-related changes in the gene expression of structural proteins in human hair follicles.

Experimental design

For the evaluation of differential gene expression in groups of different ages, 10 scalp hair follicles for every 20 healthy volunteers (10 women/10 men) below 25 years and above 50 years of age were plucked and total RNA was isolated.

Following standard reverse transcription of each RNA sample, quantitative polymerase chain reaction was performed with gene-specific primer sets for different hair keratins (Table S1). The resulting relative expression values of both groups were compared, whereby statistical significant differences were proven by a student t-test.

For extended gene expression analysis, the samples were pooled and the experiments were performed at Miltenyi Biotec (Bergisch Gladbach, Germany) using an Agilent whole-human genome microarray according to the manufacturer’s instructions. All methods are described in detail in Appendix S1 in the Supporting information.

Results

Our data show that the ageing process in follicles is associated among other things with a decline of structural proteins such as certain hair keratins and keratin-associated proteins (KAPs). Molecular analysis of keratin gene expression in hair follicles of donors above 50 years and below 25 years of age revealed striking differences between these two groups. While the expression of KRT31, KRT32, KRT36, KRT85 and KRT86 seemed to remain unaffected by ageing, we registered a statistically significant decline in gene activity of KRT33A and KRT34 in the older group of above 50 years of age compared with the younger group (Fig. 1). Remarkably, the regulated hair-specific keratins KRT33A and KRT34 belong to a group of keratins which are expressed in the upper part of the follicular cortex, representing late differentiation products within the hair-forming compartment (7).

Figure 1.Gene expression analysis of different hair keratins using a quantitative PCR method. Data show significant down-regulation of KRT33A and KRT34 in the older follicles compared to the younger ones. Statistics are given as SEM, * P < 0.05.

Furthermore, whole-human genome microarray analysis demonstrated a clear diminution of gene expression regarding certain KAPs, which represent the cross-linking network between the keratin intermediate filaments (8), in the hair follicles of older volunteers. From 39 analysed KAP genes, seven members of the family are downregulated significantly in aged hair follicles, which have been proven byanova/t-test. Thereby, the group of KAP4 genes seems to undergo the most vigorous age-related changes. Additionally, two members of two further KAP families are underrepresented in RNA samples of mature hair follicles (Fig. 2).

Figure 2.Gene expression analysis using a cDNA microarray. Data show significant down-regulation of different keratin associated proteins in the older follicles compared to the younger ones. Statistics are given as SEM, * P < 0.05.

Conclusion

The present studies enabled us to get a deeper insight into molecular events accompanying hair follicle ageing, especially the parameters related to hair fibre composition. Keratins are the most abundant structural proteins in hair (9) and the microfibrils or intermediate filaments they create are primarily responsible for hair’s mechanical properties (10). Thus, their adequate synthesis might be a prerequisite to maintain hair’s juvenescent characteristics. Here, we showed that the expression of two members of the acidic keratin family, keratin KRT33A and keratin KRT34, decrease with age. As they represent the later differentiation of the hair follicle, we speculate that this might be aetiological for the previously reported changes in hair structure during ageing (11). Besides the hair keratins, KAPs represent the other main components of the hair fibre, forming the protein matrix between the keratin microfibrils (12) and playing a crucial role in forming a strong hair shaft (8). As the KAP4 family not only represents the largest KAP family but also has been shown to be expressed predominantly in the highly differentiated portions of the middle and upper cortex and is suggested to be heavily involved in the terminal keratinization of the hair fibre cortex (13), the strong decline in gene expression therefore might influence hair shaft stability and flexibility. To evaluate whether the age-related changes in the gene expression pattern also become manifest in the ultrastructure of the hair follicle and fibre, further analysis using transmission electron microscopy as it has been proposed by Morioka (14) might be of interest. Already today, we have first evidence that the described age-related changes in the hair follicle influence the later hair fibre, as protein extracts of the hair shaft analysed by mass spectroscopy following separation by gel electrophoresis showed certain age-dependent differences in the peptide pattern (data not shown). Further studies will show whether those alterations can explain the macroscopic changes of mature hair and how they influence the mechanical behaviour.

Acknowledgements

We thank Ms. Sabine Gruedl for her technical assistance and the analysis of parts of the data sets. Mr. Olaf Holtkoetter contributes to the analysis of the microarray data sets. Mr. Guido Fuhrmann supported the sample collection and preparation of the samples. Mrs. Andrea Koerner was responsible for the analysis of hair fibres using mass spectroscopy techniques and Mr. Dirk Petersohn revised and approved the manuscript. Mrs. Melanie Giesen designed the study, analysed parts of the data sets and wrote the paper.